Virus Filtration of Liquid Factor VII Compositions

ABSTRACT

The present invention relates to a novel method for improving the viral safety of liquid Factor VII compositions, in particular those comprising active Factor VII polypeptides (a Factor VIIa polypeptide).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/349,980, filed Jan. 13, 2012 (Notice of Allowance mailed), which is acontinuation of U.S. patent application Ser. No. 12/173,475, filed Jul.15, 2008 (abandoned), which is a continuation of U.S. patent applicationSer. No. 11/439,828, filed May 23, 2006 (abandoned), which is acontinuation of International Patent Application No. PCT/DK04/53206,filed Dec. 1, 2004, which claims priority from Danish Patent ApplicationNo. PA 2003 01775, filed Dec. 1, 2003; and to U.S. Patent ApplicationNo. 60/528,763, filed Dec. 11, 2003; the contents of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a novel method for improving the viralsafety of liquid Factor VII compositions, in particular those comprisingactive Factor VII polypeptides (a Factor VIIa polypeptide).

BACKGROUND OF THE INVENTION

A variety of Factors involved in the blood clotting process have beenidentified, including Factor VII (FVII), a plasma glycoprotein.Haemostasis is initiated by the formation of a complex between TissueFactor (TF) being exposed to the circulating blood following an injuryto the vessel wall, and Factor VIIa which is present in the circulationin an amount corresponding to about 1% of the total Factor VII proteinmass. Factor VII exists in plasma mainly as a single-chain zymogen whichis cleaved by FXa into its two-chain, activated form, Factor VIIa.Recombinant activated Factor VIIa (rFVIIa) has been developed as apro-haemostatic agent. The administration of rFVIIa offers a rapid andhighly effective pro-haemostatic response in haemophilic subjects withbleedings, who cannot be treated with other coagulation Factor productsdue to antibody formation. Also bleeding in subjects with Factor VIIdeficiency or subjects having a normal coagulation system butexperiencing excessive bleeding can be treated successfully with FactorVIIa.

The purification and handling of Factor VII must be careful, due thepossibility for degradation of the molecule. Factor VII and Factor VIIa,being large molecules (approx. molecular weight 50 kD), are susceptibleto mechanical degradation by shear forces during purification andfiltration. Further, Factor VIIa is an active proteolytic enzyme thatdegrades other proteins including Factor VIIa. Degradation of FactorVIIa mainly involves cleavage in the heavy chain of Factor VIIa,particularly at amino acids no. 290 and 315 in the molecule. Finally,methionine residues in Factor VII and Factor VIIa may be oxidized.

An object of the present invention is to provide a method for theremoval or inactivation of viruses from liquid Factor VII compositionsby which method the integrity of the Factor VII constituents issubstantially preserved.

WO 96/00237 discloses a method of virus-filtration of a solution thatcontains a macromolecular, e.g. a protein such as the plasma proteinFactor IX.

WO 98/37086 discloses removal of viruses from plasma-derived proteinsolutions by nanofiltration using a membrane having an average pore sizeof 15 nm.

Tomokiyo et al., Vox Sanguinis, 2003, 84, 54-64, disclose thelarge-scale production of human plasma-derived activated Factor VIIconcentrate. The method of production involves the step ofvirus-filtration of a solution comprising inactive Factor VII.

BRIEF DESCRIPTION OF THE INVENTION

In a broad aspect, the preset invention relates to methods for theremoval and/or inactivation of viruses from Factor VII composition. Theterm “virus” as used herein means any ultramicroscopic infectious agentthat replicates itself only within cells of living hosts, ornoninfectious particles derived thereof. In one embodiment the virus isinfectious. In one embodiment the virus is a non-infectious virusparticle.

A first aspect of the present invention relates to a method for removingviruses from a liquid Factor VII composition, said method comprisingsubjecting said solution to nanofiltration using a nanofilter having apore size of at the most 80 nm.

A second aspect of the present invention relates to a method forremoving viruses from a liquid Factor VII composition, said compositioncomprising one or more Factor VII polypeptides, at least 5% of said oneor more Factor VII polypeptides being in the activated form, said methodcomprising subjecting said solution to nanofiltration using a nanofilterhaving a pore size of at the most 80 nm.

A third aspect of the invention relates to a method for removing virusesfrom a liquid Factor VII composition, said composition comprising one ormore Factor VII polypeptides, said liquid composition beingsubstantially serum-free, said method comprising subjecting saidsolution to nanofiltration using a nanofilter having a pore size of atthe most 80 nm.

A further aspect of the invention relates to a method for removingviruses from a liquid Factor VII composition, said compositioncomprising one or more Factor VII polypeptides, said method comprisingsubjecting said solution to nanofiltration using a nanofilter having apore size of at the most 80 nm, said nanofilter having a membranemanufactured from one or more materials selected from cuprammoniumregenerated cellulose, hydrophilic polyvinylidene fluoride (PVDF),composite PVDF, surface modified PVDF, and polyether sulfone.

A further aspect of the invention relates to a method for inactivatingviruses in a liquid Factor VII composition, said composition comprisingone or more Factor VII polypeptides, the method comprising the step ofcombining said composition with a detergent.

A further aspect of the invention relates to a method for high-levelelimination of the presence of active viruses in a liquid Factor VIIcomposition, the method comprising the steps of (i) inactivatingviruses, and (ii) removing viruses.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a system suitable for methods ofthe invention. The system includes a pressure tank (1) with a supply ofcompressed air, a pre-filter (2) for removing particles that wouldotherwise clog the virus filter, a pressure gauge (P), a virus filter(3), and a pool tank (4).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for removing or inactivatingviruses, including non-enveloped viruses, from a liquid Factor VIIcomposition which typically comprises a significant ratio of activatedand thereby proteolytically active Factor VII polypeptides. The methodincludes the step of subjecting the liquid Factor VII composition tonanofiltration using a nanofilter having a pore size of at the most 80nm.

The method is particularly useful for the removal of enveloped virusesas well as non-enveloped viruses such as Murine Leukemia virus(enveloped) which may be removed by filters with a pore size around 50nm, and Porcine Parvovirus (non-enveloped) which may be removed byfilters with a pore size around 20 nm.

The liquid Factor VII compositions, e.g. those comprising a significantratio of activated Factor VII polypeptides, can in principle be preparedfrom the dry Factor VII constituents, but are more typically obtainedfrom large-scale production processes, e.g. processes involvingrecombinant techniques. In such processes a cell culture supernatant istypically harvested and subsequently subjected to one or more processingsteps to obtain the desired protein, including, without limitation,centrifugation or filtration to remove cells that were not immobilizedin the carriers; affinity chromatography, hydrophobic interactionchromatography; ion-exchange chromatography; size exclusionchromatography; electrophoretic procedures (e.g., preparativeisoelectric focusing (IEF), differential solubility (e.g., ammoniumsulfate precipitation), or extraction and the like. See, generally,Scopes, Protein Purification, Springer-Verlag, New York, 1982; andProtein Purification, J. -C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989. Purification of Factor VII polypeptides mayalso involve, e.g., affinity chromatography on an anti-Factor VIIantibody column (see, e.g., Wakabayashi et al., J. Biol. Chem.261:11097, 1986; and Thim et al., Biochem. 27:7785, 1988) and activationby proteolytic cleavage, using Factor XIIa or other proteases havingtrypsin-like specificity, such as, e.g., Factor IXa, kallikrein, FactorXa, and thrombin. See, e.g., Osterud et al., Biochem. 11:2853 (1972);Thomas, U.S. Pat. No. 4,456,591; and Hedner et al., J. Clin. Invest.71:1836 (1983). Alternatively, a Factor VII polypeptide may be activatedby passing it through an ion-exchange chromatography column, such asMono Q® (Pharmacia) or the like.

The methods of the present invention are particularly useful forlarge-scale production processes. By the term “large-scale” is typicallymeant methods wherein the volume of the liquid Factor VII polypeptidecompositions is at least 100 L, such as at least 500 L, e.g. at least1000 L, or at least 5000 L. This is not to be limiting in any way, asthe present invention will also work for liquid Factor VII polypeptidecompositions of less than 100 L.

It has now been realized that nanofiltration may be applied even afterthe Factor VII polypeptide bulk has been partially or fully activated.

Thus, the methods of the invention are applicable as one of the steps ofthe overall purification process for the Factor VII polypeptide,typically one of the final steps of the purification process.

More specifically, a typical purification process starting fromharvested material from the a fermentation broth (or from human (ormammalian) plasma) can be outlined as follows:

Purification Possible stages for step virus filtration Harvest ↓ 1Capture ↓ 2 Intermediate purification ↓ 3 Polishing ↓ 4 Drug substance

The content of Factor VII polypeptide in the activated form is initially(i.e. from the harvest step) typically around 2%, and increases in thecourse of the purification process to 90% or more before the polypeptideis obtained as a drug substance.

The liquid Factor VII composition subjected to nanofiltration comprisesone or more Factor VII polypeptides in a suitable solvent. The solventis typically water or an aqueous mixture/solution, such as pure water,an aqueous buffer, a water/ethanol mixture, a water/DMSO mixture, or anaqueous salt solution, e.g. saline, a urea solution or guanidinesolution. A suitable aqueous liquid may also comprise a detergent(surfactant).

In interesting embodiments, the liquid Factor VII composition isobtained, or originates, from a cell culture supernatant, e.g. a cellculture supernatant obtained as disclosed in WO 02/29084. In oneembodiment, the liquid Factor VII composition is serum-free, i.e. freefrom animal-derived components. Thus, the cell cultures may becultivated in a medium lacking animal derived components.

An attractive variant hereof is the one where the Factor VIIpolypeptide(s) is/are produced by cell culture in CHO cells, e.g. in CHOcells in a medium free from any components of animal origin, or a mediumlacking animal-derived components and lacking proteins (“protein-free”).

The medium for CHO cells may be any commercially available protein-freeCHO medium lacking animal-derived components or an in-house producedmedium for CHO cells.

In some embodiments, the cells used in practicing the present inventionare adapted to suspension growth in medium lacking animal-derivedcomponents, such as, e.g., medium lacking serum. Such adaptationprocedures are described, e.g., in Scharfenberg, et al., Animal CellTechnology Developments towards the 21^(st) Century, E. C. Beuvery etal. (Eds.), Kluwer Academic Publishers, pp. 619-623, 1995 (BHK and CHOcells); Cruz, Biotechnol. Tech. 11:117-120, 1997 (insect cells); Keen,Cytotechnol. 17:203-211, 1995 (myeloma cells); Berg et al.,Biotechniques 14:972-978, 1993 (human kidney 293 cells). In aparticularly embodiment, the host cells are BHK 21 or CHO cells thathave been engineered to express human Factor VII or a Factor VIIpolypeptide and that have been adapted to grow in the absence of serumor animal-derived components.

In an alternative embodiment, the Factor VII polypeptide(s) is/areproduced by cell culture in the presence of bovine or fetal calf serum.

According to one aspect of the invention, a feature is that asignificant ratio, i.e. at least 5%, such as at least 7%, e.g. at least10%, of the one or more Factor VII polypeptides are in the activatedform (i.e. the bioactive, cleaved form of a Factor VII polypeptide (i.e.a Factor VIIa polypeptide)). In further embodiments, the Factor VIIapolypeptide represents 5-70%, such as 7-40%, e.g. 10-30%, of the mass ofthe one or more Factor VII polypeptides. In other embodiments, theFactor VIIa polypeptide represents 50-100%, such as 70-100%, e.g.80-100%, of the mass of the one or more Factor VII polypeptides. Instill other embodiments, the Factor VIIa polypeptide represents 20-80%,such as 30-70%, e.g. 30-60%, of the mass of the one or more Factor VIIpolypeptides.

In most embodiments, the solution comprises a Factor VII polypeptide ininactivated form as well as a bioactive Factor VIIa polypeptide, i.e.the Factor VIIa polypeptide represents less than 100% of the mass of theone or more Factor VII polypeptides. In the most typical embodiment, thecomposition comprises a(n) (activated) Factor VIIa polypeptide thatcorresponds to an (inactive) Factor VII polypeptide, i.e. the FactorVIIa polypeptide is the Factor VII polypeptide in the activated form. Inother embodiment, the Factor VIIa polypeptide is somewhat different fromthe activated form of the inactivated Factor VII polypeptide. It shouldof course be understood that the composition in particular embodimentsmay comprise more than one Factor VII polypeptide and more than oneFactor VIIa polypeptide.

The term “one or more Factor VII polypeptides” encompasses wild-typeFactor VII (i.e. a polypeptide having the amino acid sequence disclosedin U.S. Pat. No. 4,784,950), as well as variants of Factor VIIexhibiting substantially the same or improved biological activityrelative to wild-type Factor VII. The term “Factor VII” is intended toencompass Factor VII polypeptides in their uncleaved (zymogen) form, aswell as those that have been proteolytically processed to yield theirrespective bioactive forms, which may be designated Factor VIIa.Typically, Factor VII is cleaved between residues 152 and 153 to yieldFactor VIIa. The term “Factor VIIa” specifically means an activated(i.e. bioactive, cleaved) Factor VII polypeptide. Thus, “Factor VIIa” isa subgroup relative to “Factor VII”. The term “inactive Factor VII”specifically means Factor VII not being Factor VIIa.

The term “Factor VII polypeptide” also encompasses polypeptides,including variants, in which the Factor VIIa biological activity hasbeen substantially modified or somewhat reduced relative to the activityof wild-type Factor VIIa, as well as Factor VII derivatives and FactorVII conjugates. These polypeptides include, without limitation, FactorVII or Factor VIIa into which specific amino acid sequence alterationshave been introduced that modify or disrupt the bioactivity of thepolypeptide. The term “Factor VII derivative” as used herein, isintended to designate wild-type Factor VII, variants of Factor VIIexhibiting substantially the same or improved biological activityrelative to wild-type Factor VII and Factor VII-related polypeptides, inwhich one or more of the amino acids of the parent peptide have beenchemically and/or enzymatically modified, e.g. by alkylation,glycosylation, PEGylation, acylation, ester formation or amide formationor the like. This includes but is not limited to PEGylated human FactorVIIa, cysteine-PEGylated human Factor VIIa and variants thereof.Non-limiting examples of Factor VII derivatives includes GlycoPegylatedFVII derivatives as disclosed in WO 03/31464 and US Patent applicationsUS 20040043446, US 20040063911, US 20040142856, US 20040137557, and US20040132640 (Neose Technologies, Inc.); FVII conjugates as disclosed inWO 01/04287, US patent application 20030165996, WO 01/58935, WO 03/93465(Maxygen ApS) and WO 02/02764, US patent application 20030211094(University of Minnesota).

The term “PEGylated human Factor VIIa” means human Factor VIIa, having aPEG molecule conjugated to a human Factor VIIa polypeptide. It is to beunderstood, that the PEG molecule may be attached to any part of theFactor VIIa polypeptide including any amino acid residue or carbohydratemoiety of the Factor VIIa polypeptide. The term “cysteine-PEGylatedhuman Factor VIIa” means Factor VIIa having a PEG molecule conjugated toa sulfhydryl group of a cysteine introduced in human Factor VIIa.

The biological activity of Factor VIIa in blood clotting derives fromits ability to (i) bind to Tissue Factor (TF) and (ii) catalyze theproteolytic cleavage of Factor IX or Factor X to produce activatedFactor IX or X (Factor IXa or Xa, respectively).

For the purposes of the invention, biological activity of Factor VIIpolypeptides (“Factor VII biological activity”) may be quantified bymeasuring the ability of a preparation to promote blood clotting usingFactor VII-deficient plasma and thromboplastin, as described, e.g., inU.S. Pat. No. 5,997,864 or WO 92/15686. In this assay, biologicalactivity is expressed as the reduction in clotting time relative to acontrol sample and is converted to “Factor VII units” by comparison witha pooled human serum standard containing 1 unit/mL Factor VII activity.Alternatively, Factor VIIa biological activity may be quantified by (i)measuring the ability of Factor VIIa (or the Factor VII polypeptide) toproduce activated Factor X (Factor Xa) in a system comprising TFembedded in a lipid membrane and Factor X. (Persson et al., 3. Biol.Chem. 272:19919-19924, 1997); (ii) measuring Factor X hydrolysis in anaqueous system (“In Vitro Proteolysis Assay”, see below); (iii)measuring the physical binding of Factor VIIa (or the Factor VIIpolypeptide) to TF using an instrument based on surface plasmonresonance (Persson, FEBS Letts. 413:359-363, 1997); (iv) measuringhydrolysis of a synthetic substrate by Factor VIIa (or a Factor VIIpolypeptide) (“In Vitro Hydrolysis Assay”, see below); or (v) measuringgeneration of thrombin in a TF-independent in vitro system.

Factor VII variants having substantially the same or improved biologicalactivity relative to wild-type Factor VIIa encompass those that exhibitat least about 25%, such as at least about 50%, such as at least about75%, such as at least about 90% of the specific activity of Factor VIIathat has been produced in the same cell type, when tested in one or moreof a clotting assay, proteolysis assay, or TF binding assay as describedabove. In one embodiment the biological activity is more than 80% of thebiological activity of recombinant wild type human Factor VIIa. Inanother embodiment the biological activity is more than 90% of thebiological activity of recombinant wild type human Factor VIIa. In afurther embodiment the biological activity is more than 100% of thebiological activity of recombinant wild type human Factor VIIa. In afurther embodiment the biological activity is more than 120% of thebiological activity of recombinant wild type human Factor VIIa. In afurther embodiment the biological activity is more than 200% of thebiological activity of recombinant wild type human Factor VIIa. In afurther embodiment the biological activity is more than 400% of thebiological activity of recombinant wild type human Factor VIIa.

Factor VII variants having substantially reduced biological activityrelative to wild-type Factor VIIa are those that exhibit less than about25%, such as less than about 10%, such as less than about 5%, such asless than about 1% of the specific activity of wild-type Factor VIIathat has been produced in the same cell type when tested in one or moreof a clotting assay, proteolysis assay, or TF binding assay as describedabove. Factor VII variants having a substantially modified biologicalactivity relative to wild-type Factor VII include, without limitation,Factor VII variants that exhibit TF-independent Factor X proteolyticactivity and those that bind TF but do not cleave Factor X.

Variants of Factor VII, whether exhibiting substantially the same orbetter bioactivity than wild-type Factor VII, or, alternatively,exhibiting substantially modified or reduced bioactivity relative towild-type Factor VII, include, without limitation, polypeptides havingan amino acid sequence that differs from the sequence of wild-typeFactor VII by insertion, deletion, or substitution of one or more aminoacids.

Non-limiting examples of Factor VII variants having substantially thesame biological activity as wild-type Factor VII include S52A-FVIIa,S60A-FVIIa (Lino et al., Arch. Biochem. Biophys. 352: 182-192, 1998);Factor VIIa variants exhibiting increased proteolytic stability asdisclosed in U.S. Pat. No. 5,580,560; Factor VIIa that has beenproteolytically cleaved between residues 290 and 291 or between residues315 and 316 (Mollerup et al., Biotechnol. Bioeng. 48:501-505, 1995);oxidized forms of Factor VIIa (Kornfelt et al., Arch. Biochem. Biophys.363:43-54, 1999); Factor VII variants as disclosed in PCT/DK02/00189;and Factor VII variants exhibiting increased proteolytic stability asdisclosed in WO 02/38162 (Scripps Research Institute); Factor VIIvariants having a modified Gla-domain and exhibiting an enhancedmembrane binding as disclosed in WO 99/20767, U.S. Pat. No. 6,017,882and U.S. Pat. No.6,747,003, US patent application 20030100506(University of Minnesota) and WO 00/66753, US patent applications US20010018414, US 2004220106, and US 200131005, U.S. Pat. No. 6,762,286and U.S. Pat. No. 6,693,075 (University of Minnesota); and Factor VIIvariants as disclosed in WO 01/58935, U.S. Pat. No. 6,806,063, US patentapplication 20030096338 (Maxygen ApS), WO 03/93465 (Maxygen ApS) and WO04/029091 (Maxygen ApS).

Non-limiting examples of Factor VII variants having increased biologicalactivity compared to wild-type Factor VIIa include Factor VII variantsas disclosed in WO 01/83725, WO 02/22776, WO 02/077218, WO 03/27147, WO03/37932; WO 02/38162 (Scripps Research Institute); and Factor VIIavariants with enhanced activity as disclosed in JP 2001061479(Chemo-Sero-Therapeutic Res Inst.).

Non-limiting examples of Factor VII variants having substantiallyreduced or modified biological activity relative to wild-type Factor VIIinclude R152E-FVIIa (Wildgoose et al., Biochem 29:3413-3420, 1990),S344A-FVIIa (Kazama et al., 3. Biol. Chem. 270:66-72, 1995), FFR-FVIIa(Hoist et al., Eur. J. Vasc. Endovasc. Surg. 15:515-520, 1998), andFactor VIIa lacking the Gla domain, (Nicolaisen et al., FEBS Letts.317:245-249, 1993).

Explicit examples of Factor VII polypeptides include, withoutlimitation, wild-type Factor VII, L305V-FVII, L305V/M306D/D309S-FVII,L305I-FVII, L305T-FVII, F374P-FVII, V158T/M298Q-FVII,V158D/E296V/M298Q-FVII, K337A-FVII, M298Q-FVII, V158D/M298Q-FVII,L305V/K337A-FVII, V158D/E296V/M298Q/L305V-FVII,V158D/E296V/M298Q/K337A-FVII, V158D/E296V/M298Q/L305V/K337A-FVII,K157A-FVII, E296V-FVII, E296V/M298Q-FVII, V158D/E296V-FVII,V158D/M298K-FVII, and S336G-FVII, L305V/K337A-FVII, L305V/V158D-FVII,L305V/E296V-FVII, L305V/M298Q-FVII, L305V/V158T-FVII,L305V/K337A/V158T-FVII, L305V/K337A/M298Q-FVII, L305V/K337A/E296V-FVII,L305V/K337A/V158D-FVII, L305V/V158D/M298Q-FVII, L305V/V158D/E296V-FVII,L305V/V158T/M298Q-FVII, L305V/V158T/E296V-FVII, L305V/E296V/M298Q-FVII,L305V/V158D/E296V/M298Q-FVII, L305V/V158T/E296V/M298Q-FVII,L305V/V158T/K337A/M298Q-FVII, L305V/V158T/E296V/K337A-FVII,L305V/V158D/K337A/M298Q-FVII, L305V/V158D/E296V/K337A-FVII,L305V/V158D/E296V/M298Q/K337A-FVII, L305V/V158T/E296V/M298Q/K337A-FVII,S314E/K316H-FVII, S314E/K316Q-FVII, S314E/L305V-FVII, S314E/K337A-FVII,S314E/V158D-FVII, S314E/E296V-FVII, S314E/M298Q-FVII, S314E/V158T-FVII,K316H/L305V-FVII, K316H/K337A-FVII, K316H/V158D-FVII, K316H/E296V-FVII,K316H/M298Q-FVII, K316H/V158T-FVII, K316Q/L305V-FVII, K316Q/K337A-FVII,K316Q/V158D-FVII, K316Q/E296V-FVII, K316Q/M298Q-FVII, K316Q/V158T-FVII,S314E/L305V/K337A-FVII, S314E/L305V/V158D-FVII, S314E/L305V/E296V-FVII,S314E/L305V/M298Q-FVII, S314E/L305V/V158T-FVII,S314E/L305V/K337A/V158T-FVII, S314E/L305V/K337A/M298Q-FVII,S314E/L305V/K337A/E296V-FVII, S314E/L305V/K337A/V158D-FVII,S314E/L305V/V158D/M298Q-FVII, S314E/L305V/V158D/E296V-FVII,S314E/L305V/V158T/M298Q-FVII, S314E/L305V/V158T/E296V-FVII,S314E/L305V/E296V/M298Q-FVII, S314E/L305V/V158D/E296V/M298Q-FVII,S314E/L305V/V158T/E296V/M298Q-FVII, S314E/L305V/V158T/K337A/M298Q-FVII,S314E/L305V/V158T/E296V/K337A-FVII, S314E/L305V/V158D/K337A/M298Q-FVII,S314E/L305V/V158D/E296V/K337A-FVII,S314E/L305V/V158D/E296V/M298Q/K337A-FVII,S314E/L305V/V158T/E296V/M298Q/K337A-FVII, K316H/L305V/K337A-FVII,K316H/L305V/V158D-FVII, K316H/L305V/E296V-FVII, K316H/L305V/M298Q-FVII,K316H/L305V/V158T-FVII, K316H/L305V/K337A/V158T-FVII,K316H/L305V/K337A/M298Q-FVII, K316H/L305V/K337A/E296V-FVII,K316H/L305V/K337A/V158D-FVII, K316H/L305V/V158D/M298Q-FVII,K316H/L305V/V158D/E296V-FVII, K316H/L305V/V158T/M298Q-FVII,K316H/L305V/V158T/E296V-FVII, K316H/L305V/E296V/M298Q-FVII,K316H/L305V/V158D/E296V/M298Q-FVII, K316H/L305V/V158T/E296V/M298Q-FVII,K316H/L305V/V158T/K337A/M298Q-FVII, K316H/L305V/V158T/E296V/K337A-FVII,K316H/L305V/V158D/K337A/M298Q-FVII, K316H/L305V/V158D/E296V/K337A -FVII,K316H/L305V/V158D/E296V/M298Q/K337A-FVII,K316H/L305V/V158T/E296V/M298Q/K337A-FVII, K316Q/L305V/K337A-FVII,K316Q/L305V/V158D-FVII, K316Q/L305V/E296V-FVII, K316Q/L305V/M298Q-FVII,K316Q/L305V/V158T-FVII, K316Q/L305V/K337A/V158T-FVII,K316Q/L305V/K337A/M298Q-FVII, K316Q/L305V/K337A/E296V-FVII,K316Q/L305V/K337A/V158D-FVII, K316Q/L305V/V158D/M298Q-FVII,K316Q/L305V/V158D/E296V-FVII, K316Q/L305V/V158T/M298Q-FVII,K316Q/L305V/V158T/E296V-FVII, K316Q/L305V/E296V/M298Q-FVII,K316Q/L305V/V158D/E296V/M298Q-FVII, K316Q/L305V/V158T/E296V/M298Q-FVII,K316Q/L305V/V158T/K337A/M298Q-FVII, K316Q/L305V/V158T/E296V/K337A-FVII,K316Q/L305V/V158D/K337A/M298Q-FVII, K316Q/L305V/V158D/E296V/K337A -FVII,K316Q/L305V/V158D/E296V/M298Q/K337A-FVII,K316Q/L305V/V158T/E296V/M298Q/K337A-FVII, F374Y/K337A-FVII,F374Y/V158D-FVII, F374Y/E296V-FVII, F374Y/M298Q-FVII, F374Y/V158T-FVII,F374Y/S314E-FVII, F374Y/L305V-FVII, F374Y/L305V/K337A-FVII,F374Y/L305V/V158D-FVII, F374Y/L305V/E296V-FVII, F374Y/L305V/M298Q-FVII,F374Y/L305V/V158T-FVII, F374Y/L305V/S314E-FVII, F374Y/K337A/S314E-FVII,F374Y/K337A/V158T-FVII, F374Y/K337A/M298Q-FVII, F374Y/K337A/E296V-FVII,F374Y/K337A/V158D-FVII, F374Y/V158D/S314E-FVII, F374Y/V158D/M298Q-FVII,F374Y/V158D/E296V-FVII, F374Y/V158T/S314E-FVII, F374Y/V158T/M298Q-FVII,F374Y/V158T/E296V-FVII, F374Y/E296V/S314E-FVII, F374Y/S314E/M298Q-FVII,F374Y/E296V/M298Q-FVII, F374Y/L305V/K337A/V158D-FVII,F374Y/L305V/K337A/E296V-FVII, F374Y/L305V/K337A/M298Q-FVII,F374Y/L305V/K337A/V158T-FVII, F374Y/L305V/K337A/S314E-FVII,F374Y/L305V/V158D/E296V-FVII, F374Y/L305V/V158D/M298Q-FVII,F374Y/L305V/V158D/S314E-FVII, F374Y/L305V/E296V/M298Q-FVII,F374Y/L305V/E296V/V158T-FVII, F374Y/L305V/E296V/S314E-FVII,F374Y/L305V/M298Q/V158T-FVII, F374Y/L305V/M298Q/S314E-FVII,F374Y/L305V/V158T/S314E-FVII, F374Y/K337A/S314E/V158T-FVII,F374Y/K337A/S314E/M298Q-FVII, F374Y/K337A/S314E/E296V-FVII,F374Y/K337A/S314E/V158D-FVII, F374Y/K337A/V158T/M298Q-FVII,F374Y/K337A/V158T/E296V-FVII, F374Y/K337A/M298Q/E296V-FVII,F374Y/K337A/M298Q/V158D-FVII, F374Y/K337A/E296V/V158D-FVII,F374Y/V158D/S314E/M298Q-FVII, F374Y/V158D/S314E/E296V-FVII,F374Y/V158D/M298Q/E296V-FVII, F374Y/V158T/S314E/E296V-FVII,F374Y/V158T/S314E/M298Q-FVII, F374Y/V158T/M298Q/E296V-FVII,F374Y/E296V/S314E/M298Q-FVII, F374Y/L305V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/K337A/S314E-FVII, F374Y/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A -FVII, F374Y/L305V/E296V/M298Q/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A-FVII, F374Y/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/V158D/K337A/S314E-FVII, F374Y/V158D/M298Q/K337A/S314E-FVII,F374Y/V158D/E296V/K337A/S314E-FVII, F374Y/L305V/V158D/E296V/M298Q-FVII,F374Y/L305V/V158D/M298Q/K337A-FVII, F374Y/L305V/V158D/E296V/K337A-FVII,F374Y/L305V/V158D/M298Q/S314E-FVII, F374Y/L305V/V158D/E296V/S314E-FVII,F374Y/V158T/E296V/M298Q/K337A-FVII, F374Y/V158T/E296V/M298Q/S314E-FVII,F374Y/L305V/V158T/K337A/S314E-FVII, F374Y/V158T/M298Q/K337A/S314E-FVII,F374Y/V158T/E296V/K337A/S314E-FVII, F374Y/L305V/V158T/E296V/M298Q-FVII,F374Y/L305V/V158T/M298Q/K337A-FVII, F374Y/L305V/V158T/E296V/K337A-FVII,F374Y/L305V/V158T/M298Q/S314E-FVII, F374Y/L305V/V158T/E296V/S314E-FVII,F374Y/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/V158D/E296V/M298Q/K337A/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/S314E-FVII,F374Y/L305V/E296V/M298Q/V158T/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T-FVII,F374Y/L305V/E296V/K337A/V158T/S314E-FVII,F374Y/L305V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A-FVII,F374Y/L305V/V158D/E296V/K337A/S314E-FVII,F374Y/L305V/V158D/M298Q/K337A/S314E-FVII,F374Y/L305V/E296V/M298Q/K337A/V158T/S314E-FVII,F374Y/L305V/V158D/E296V/M298Q/K337A/S314E-FVII, S52A-Factor VII,S60A-Factor VII; R152E-Factor VII, S344A-Factor VII, Factor VIIa lackingthe Gla domain; and P11Q/K33E-FVII, T106N-FVII, K143N/N145T-FVII,V253N-FVII, R290N/A292T-FVII, G291N-FVII, R315N/V317T-FVII,K143N/N145T/R315N/V317T-FVII; and Factor VII having substitutions,additions or deletions in the amino acid sequence from 233Thr to 240Asn,Factor VII having substitutions, additions or deletions in the aminoacid sequence from 304Arg to 329Cys.

In some embodiments, the Factor VIIa polypeptide is human Factor VIIa(hFVIIa), such as recombinantly made human Factor VIIa (rhFVIIa). Inother embodiments, the one or more Factor VII polypeptides comprise aFactor VII sequence variant. In some embodiments, the one or more FactorVII polypeptides have a glycosylation different from wild-type FactorVII.

Nanofiltration

The liquid Factor VII composition is subjected to nanofiltration using ananofilter having a pore size of at the most 80 nm. The pore size of thenanofilter is more particularly at the most 50 nm, e.g. at the most 30nm, such as in the range of 10-30 nm.

The term “pore size” typically means the size of the smallest virusesthat are withheld by the filter.

Examples of suitable commercially available nanofilters are AsahiPlanove 15 N, Asahi Planove 20 N, Asahi Planova 35 N, and Asahi Planova75 N, all from Asahi Chemical, Tokyo, Japan; Millipore NFR, MilliporeNFP, Millipore Viresolve 70, and Millipore Viresolve 180, all fromMillipore; and Pall DV20, Pall DV 50, Pall Omega VR 100 K; and BembergMicroporous Membrane-15 nm (BMM-15).

The nanofilter membrane may, e.g., be manufactured from one or morematerials selected from cuprammonium regenerated cellulose, hydrophilicpolyvinylidene fluoride (PVDF), composite PVDF, surface modified PVDF,polyether sulfone and similar materials. In one embodiment, the materialis selected from polyvinylidene fluoride-based materials and polyethersulfone-based materials.

The nanofiltration may be conducted by in the tangential filtration modeor in the dead-end filtration mode as will be understood by the skilledartisan. In one embodiment, the nanofiltration is conducted in thedead-end filtration mode.

The pH value of the liquid Factor VII composition upon nanofiltration isnot considered particularly critical. Thus, the pH value is normallygiven by in view of the conditions applied in the process stepsimmediately preceding the nanofiltration step. In some embodiments, thepH value is adjusted so that the liquid composition has a pH of in therange of 5.5-10, such as in the range of 7.0-9.5, e.g. in the range of7.6-9.4, such as in the range of 7.7-9.3, e.g. in the range of 8.0-9.0or in the range of 8.3-8.7. In one embodiment the pH is in the range of5-7. In one embodiment the pH is higher than 9.5, such as in the rangeof 9.5-10.

Furthermore, the concentration of the Factor VII polypeptide in theliquid composition is typically also given by the preceding processsteps, but will normally lie in the range of 0.01-5 mg/mL, such as inthe range of 0.05-2.0 mg/mL.

The nanofiltration process may be conducted using a filtration system asillustrated in FIG. 1.

The process may be conducted as in the following illustrative example:The pressure tank (1) is filled with water for injection (WFI), and thepressure in the tank is raised to 3.5 bars before the virus filter (3),and the filter is flushed for 10 minutes. The pressure is reduced to 2bars and the virus filter (3) is flushed for another 10 minutes. Thepressure tank (1) is emptied from WFI and the process is optionallyrepeated with a buffer before the liquid Factor VII composition isfilled into the pressure tank (1). The pressure is raised to 2 bars andis kept substantially constant during the filtration. The virus filter(3) may subsequently be tested for integrity by standard procedures.

The filtrate is collected in a pool tank and can further be processed inorder to obtain a pharmaceutical composition comprising a Factor VIIapolypeptide as a drug substance.

This being said, it is typically advantageous to apply a pre-filtrationstep before the nanofiltration step in order to remove larger particles,aggregates, etc. that would otherwise cause the nanofilter to becomeclogged. Such a pre-filter typically has a pore size of at in the rangeof 0.05-0.5 μm. In one embodiment the pre-filter is Millipore NFRfilter.

Alternatively to using air pressure, a liquid pump placed after thepressure tank may provide the necessary pressure for the filtration.

If the nanofiltered liquid Factor VII composition comprises inactiveFactor VII polypeptides, the composition may subsequently be subjectedto an activation step, e.g. as described in Bjorn. S. & Thim, L. Res.Disclosures (1986) 269, 564-565, Pedersen, A.H. & al., Biochemistry(1989), 28, 9331-9336, and Tomokiyo, K. & al., Vox Sang. 84, 54-64(2003).

Further processing of the composition and final formulation as apharmaceutical product may be conducted as disclosed in Jurlander, B. &al., Seminars in Thrombosis and Hemostasis 27, 4, 373-383 (2001).

Nanofiltration of Serum-Free Liquid Factor VII Polypeptide Compositions

One separate aspect of the invention, which may include some or all ofthe above characteristics, relates to a method for removing viruses froma liquid Factor VII composition, said composition comprising one or moreFactor VII polypeptides, said liquid composition being substantiallyserum-free, said method comprising subjecting said solution tonanofiltration using a nanofilter having a pore size of at the most 80nm.

An attractive variant hereof is the one where the Factor VIIpolypeptide(s) is/are produced by cell culture in CHO cells, e.g. in CHOcells in a medium free from any components of animal origin.

This aspect of the invention is not particularly limited to liquidFactor VII compositions in which a certain proportion of the Factor VIIpolypeptide(s) is/are in the activated form. However, the conditionsmentioned above for the first aspect of the invention also applies forthis, the second aspect of the invention, mutatis mutandis.

Nanofiltration of Liquid Factor VII Polypeptide Compositions ViaParticular Filters

Another separate aspect of the invention, which may include some or allof the above characteristics, relates to a method for removing virusesfrom a liquid Factor VII composition, said composition comprising one ormore Factor VII polypeptides, said method comprising subjecting saidsolution to nanofiltration using a nanofilter having a pore size of atthe most 80 nm, said nanofilter having a membrane manufactured from oneor more materials selected from cuprammonium regenerated cellulose,hydrophilic polyvinylidene fluoride (PVDF), composite PVDF, surfacemodified PVDF, and polyether sulfone.

In one embodiment, the material is selected from polyvinylidenefluoride-based materials and polyether sulfone-based materials.

This aspect of the invention is not particularly limited to liquidFactor VII compositions in which a certain proportion of the Factor VIIpolypeptide(s) is/are in the activated form. However, the conditionsmentioned above for the first aspect of the invention also applies forthis, the third aspect of the invention, mutatis mutandis.

Virus Inactivation by Addition a Detergent

In another aspect, the present invention also relates to a method forinactivating viruses in a liquid Factor VII composition, saidcomposition comprising one or more Factor VII polypeptides, the methodcomprising the step of combining said composition with a detergent.

In some embodiments, the detergent is selected from non-ionic detergentssuch as those selected from octylphenoxy polyethoxyethanol,polysorbates, poloxamers, polyoxyethylene alkyl ethers,polyethylene/polypropylene block co-polymers, polyethyleneglycol (PEG),polyoxyethylene stearates, and polyoxyethylene castor oils. Illustrativeexamples hereof are non-ionic detergents are Triton X-100, Tween®,polysorbate 20, polysorbate 60, polysorbate 80, Brij-35 (polyoxyethylenedodecyl ether), poloxamer 188, poloxamer 407, PEG8000, Pluronic®polyols, polyoxy-23-lauryl ether, Myrj 49, and Cremophor A.

A particularly useful detergent is a octylphenoxy polyethoxyethanol ofthe formula p-((CH₃)₃CH₂C(CH₂)₂)—C₆H₄—O—(CH₂CH₂O)_(n)—H wherein n is inthe range of 5-15, in particular one where n is 9-10, such the detergentTriton X-100.

In one embodiment, the detergent is combined with the liquid Factor VIIcomposition to obtain a concentration of the detergent in thecomposition of in the range of 0.01-0.5% by weight, such as in the rangeof 0.05-0.4% by weight, such as in the range of 0.05-0.3% by weight,such as in the range of 0.05-0.2% by weight, such as in the range of0.05-0.1% by weight.

In a further embodiment, the detergent is combined with the compositionat a temperature of in the range of 2-12° C., such as in the range of2-9° C.

For most purposes, it is found undesirable to include atrialkylphosphate detergent, thus, the detergent may be substantiallyfree of trialkylphosphate solvents such as tri(n-butyl) phosphate.

In one particular embodiment, the method comprises the steps ofcombining the Factor VII polypeptide composition with Triton X-100 to aconcentration of 0.05-0.2% by weight at a temperature in the range of2-9° C., with the proviso that detergent is substantially free oftrialkylphosphate solvents such as tri(n-butyl)phosphate.

This aspect of the invention is not particularly limited to liquidFactor VII compositions in which a certain proportion of the Factor VIIpolypeptide(s) is/are in the activated form. However, the conditionsmentioned above for the first aspect of the invention also applies forthis, the fourth aspect of the invention, mutatis mutandis.

Combination of Virus Inactivation Steps

In a still further aspect, the present invention relates to a method forhigh-level elimination of the presence of active viruses in a liquidFactor VII composition, the method comprising the steps of (i)inactivating viruses by the method defined under “Virus inactivation byaddition a detergent”, and (ii) removing viruses by the any of themethods defined herein under “Nanofiltration”, in any order.

In one embodiment, the step of inactivating viruses precedes the step ofremoving viruses.

Even though the individual steps are believed to be sufficient for thepurpose of eliminating the presence of active viruses, the two methodscan be considered as at least partially “orthogonal” in the sense thatcertain viruses may be more difficult to eliminate by one of themethods, whereas the same of the certain viruses can more easily beeliminated by the other method, and vice versa. Thus, combination of thetwo methods will provide an even higher level of safety for the patientfor which the Factor VII polypeptide is intended, and not the least forthe medical doctor prescribing the Factor VII polypeptide medicament,and for the regulatory authorities approving the medicament. Thus, thecombination of the two methods may have a high commercial value.

As described above the present invention relates to the removal orinactivation of virus particles. The reduction of the amount of virusparticles at a particular process step is usually described in log-units(log 10 logarithm, or log₁₀) , wherein the reduction factor iscalculated as the amount of virus particles after the step relative tothe amount of virus particles before the process step.

E.g. if 10⁶ virus particles are found before a step and 10² are foundafter the step, the reduction is 10⁴, or 4 log₁₀.

The total reduction of virus particles from the complete process isdescribed in the same way and may be calculated by addition of the virusclearance from each step in the process, the word “clearance” meaningboth removal of virus and inactivation of virus

For a specific virus clearance step to be effective, it is preferred tohave a virus reduction of at least 4 log₁₀.

In one embodiment of the present invention, a filtration step reducesthe amount of virus particles with at least about 4 log₁₀. In oneembodiment of the present invention, a filtration step reduces theamount of virus particles with at least about 5 log₁₀.

In one embodiment of the present invention, a step of combining saidFVII composition with a detergent inactivates at least about 4 log₁₀ ofvirus. In one embodiment of the present invention, a step of combiningsaid FVII composition with a detergent inactivates at least about 5log₁₀ of virus.

The determination of the amount of virus particles is known to theperson skilled in the art and may be measured in standard TCID₅₀ assays(Tissue culture infectious dose 50% endpoint per mL), plaque assays orPCR assays. TCID₅₀ and plaque assays may be used to measure theconcentration of infectious particles, whereas the PCR assays may beused to measure both infectious and non-infectious inactivated virusparticles

Embodiments of the Present Invention:

1. A method for removing viruses from a liquid Factor VII composition,said composition comprising one or more Factor VII polypeptides, atleast 5% of said one or more Factor VII polypeptides being in theactivated form, said method comprising subjecting said solution tonanofiltration using a nanofilter having a pore size of at the most 80nm.2. The method according to embodiment 1, wherein as at least 7%, e.g. atleast 10%, of the one or more Factor VII polypeptides are in theactivated form.3. The method according to embodiment 1, wherein the activated form ofthe Factor VII polypeptide represents 5-70%, such as 7-40%, e.g. 10-30%,of the mass of the one or more Factor VII polypeptides.4. The method according to embodiment 1, wherein the activated form ofthe Factor VII polypeptide represents 50-100%, such as 70-100%, e.g.80-100%, of the mass of the one or more Factor VII polypeptides.5. The method according to embodiment 1, wherein the activated form ofthe Factor VII polypeptide represents 20-80%, such as 30-70%, e.g.30-60%, of the mass of the one or more Factor VII polypeptides.6. The method according to any of the preceding embodiments, wherein theliquid composition has a pH of in the range of 7.0-9.5, e.g. in therange of 7.6-9.4, such as in the range of 7.7-9.3, e.g. in the range of8.0-9.0 or in the range of 8.3-8.7.7. The method according to any of the preceding embodiments, wherein theconcentration of the Factor VII polypeptide(s) in the liquid compositionis in the range of 0.01-5 mg/mL, such as in the range of 0.05-2.0 mg/mL.8. The method according to any of the preceding embodiments, wherein thepore size of the nanofilter is at the most 50 nm, e.g. at the most 30nm, such as in the range of 10-30 nm.9. The method according to any of the preceding embodiments, wherein themembrane of the nanofilter is manufactured from one or more materialsselected from cuprammonium regenerated cellulose, hydrophilicpolyvinylidene fluoride (PVDF), composite PVDF, surface modified PVDF,and polyether sulfone.10. The method according to any of the preceding embodiments, whereinthe liquid Factor VII composition is obtained, or originates, from acell culture supernatant.11. The method according to any of the preceding embodiments, whereinthe liquid composition is substantially serum-free.12. The method according to any of the embodiments 1-10, wherein theFactor VII polypeptide(s) is/are produced by cell culture in thepresence of bovine or foetal calf serum.13. The method according to any of the preceding embodiments, whereinthe Factor VII polypeptide(s) is/are produced by cell culture in CHOcells.14. The method according to embodiment 13, wherein the Factor VIIpolypeptide(s) is/are produced by cell culture in CHO cells, in a mediumfree from any components of animal origin.15. A method for removing viruses from a liquid Factor VII composition,said composition comprising one or more Factor VII polypeptides, saidliquid composition being substantially serum-free, said methodcomprising subjecting said solution to nanofiltration using a nanofilterhaving a pore size of at the most 80 nm.16. The method according to embodiment 15, wherein the liquid Factor VIIcomposition is obtained, or originates, from a cell culture supernatant.17. The method according to any of the embodiments 15-16, wherein theFactor VII polypeptide(s) is/are produced by cell culture in CHO cells.18. The method according to embodiment 17, wherein the Factor VIIpolypeptide(s) is/are produced by cell culture in CHO cells, in a mediumfree from any components of animal origin.19. The method according to any of the embodiment 15-18, wherein atleast 5% of said one or more Factor VII polypeptides are in theactivated form.20. The method according to any of the embodiments 15-19, wherein theliquid composition has a pH of in the range of 7.0-9.5, e.g. in therange of 7.6-9.4, such as in the range of 7.7-9.3, e.g. in the range of8.0-9.0 or in the range of 8.3-8.7.21. The method according to any of the embodiments 15-20, wherein theconcentration of the Factor VII polypeptide(s) in the liquid compositionis in the range of 0.01-5 mg/mL, such as in the range of 0.05-2.0 mg/mL.22. The method according to any of the embodiments 15-21, wherein thepore size of the nanofilter is at the most 50 nm, e.g. at the most 30nm, such as in the range of 10-30 nm.23. The method according to any of the embodiments 15-22, wherein themembrane of the nanofilter is manufactured from one or more materialsselected from cuprammonium regenerated cellulose, hydrophilicpolyvinylidene fluoride (PVDF), composite PVDF, surface modified PVDF,and polyether sulfone.24. A method for removing viruses from a liquid Factor VII composition,said composition comprising one or more Factor VII polypeptides, saidmethod comprising subjecting said solution to nanofiltration using ananofilter having a pore size of at the most 80 nm, said nanofilterhaving a membrane manufactured from one or more materials selected fromcuprammonium regenerated cellulose, hydrophilic polyvinylidene fluoride(PVDF), composite PVDF, surface modified PVDF, and polyether sulfone.25. The method according to embodiment 24, wherein the material isselected from polyvinylidene fluoride-based materials and polyethersulfone-based materials.26. A method according to any of the embodiments 24-25, wherein at least5% of the one or more Factor VII polypeptides is/are in the activatedform.27. The method according to any of the embodiments 24-26, wherein theliquid composition has a pH of in the range of 7.0-9.5, e.g. in therange of 7.6-9.4, such as in the range of 7.7-9.3, e.g. in the range of8.0-9.0 or in the range of 8.3-8.7.28. The method according to any of the embodiments 24-27, wherein theconcentration of the Factor VII polypeptide(s) in the liquid compositionis in the range of 0.01-5 mg/mL, such as in the range of 0.05-2.0 mg/mL.29. The method according to any of the embodiments 24-28, wherein thepore size of the nanofilter is at the most 50 nm, e.g. at the most 30nm, such as in the range of 10-30 nm.30. The method according to any of the embodiments 24-29, wherein theliquid Factor VII composition is obtained, or originates, from a cellculture supernatant.31. The method according to any of the embodiments 24-30, wherein theliquid composition is substantially serum-free.32. The method according to any of the embodiments 24-31, wherein theFactor VII polypeptide(s) is/are produced by cell culture in thepresence of bovine or foetal calf serum.33. The method according to any of the embodiments 24-32, wherein theFactor VII polypeptide(s) is/are produced by cell culture in CHO cells.34. The method according to embodiment 33, wherein the Factor VIIpolypeptide(s) is/are produced by cell culture in CHO cells, in a mediumfree from any components of animal origin.35. A method for inactivating viruses in a liquid Factor VIIcomposition, said composition comprising one or more Factor VIIpolypeptides, the method comprising the step of combining saidcomposition with a detergent.36. The method according to embodiment 35, wherein the detergent is aoctylphenoxy polyethoxyethanol of the formulap-((CH₃)₃CH₂C(CH₂)₂)—C₆H₄—O—(CH₂CH₂O)_(n)—H wherein n is in the range of5-15.37. The method according to embodiment 36, wherein the detergent is onewhere n is 9-10, such as Triton X-100.38. The method according to embodiment 35, wherein the detergent isselected from the list consisting of Tween®, polysorbate 20, polysorbate60, and polysorbate 80.39. The method according to any of the embodiment 35-38, wherein thedetergent is combined with the liquid Factor VII composition to obtain aconcentration of the detergent in the composition of in the range of0.01-0.3% by weight, such as in the range of 0.05-0.2% by weight.40. The method according to any of the embodiments 35-39, wherein thedetergent is combined with the composition at a temperature of in therange of 2-12° C., such as in the range of 2-9° C.41. The method according to any of the embodiments 35-40, wherein thedetergent is substantially free of trialkylphosphate solvents such astri(n-butyl)phosphate.42. A method for high-level elimination of the presence of activeviruses in a liquid Factor VII composition, the method comprising thesteps of (i) inactivating viruses by the method defined in any of theembodiments 35-41, and (ii) removing viruses by the any of the methodsdefined in any of the embodiments 1-35, in any order.43. The method according to embodiment 42, wherein the step ofinactivating viruses precedes the step of removing viruses.

EXAMPLES Example 1 Serum-Free Production of Factor VII

The following experiment was performed to produce Factor VII in largepilot-scale culture.

A CHO K1 cell line transformed with a Factor VII-encoding plasmid wasadapted to growth in suspension culture in a medium free of animalderived components. A bank of the adapted cells was frozen. Cells fromthe bank were propagated in spinner bottles in suspension culture inmedium free of animal derived components. As the cell number increased,the volume was gradually increased by addition of new medium. When thevolume had reached 4 L, and the cell number had reached≈0.8*10⁶/ml, thecontents of the spinner bottles were transferred into a 50 L stirredtank reactor (seed reactor). As the cell number increased in the 50 Lreactor, the volume was gradually increased by addition of new medium.When the volume had reached 50 L, and the cell number hadreached≈1×10⁶/ml, the contents of the 50 L reactor were transferred intoa 500 L stirred tank reactor (production reactor). The 500 L reactorcontained macroporous Cytopore 1 carriers (Amersham Biosciences) withinwhich the cells became immobilized within 24 hours after inoculation.The volume in the 500 L reactor was gradually increased by addition ofnew medium as the cell number increased. When the volume had reached 450L, and the cell number had reached≈2×10⁶/ml, the production phase wasinitiated and a medium change was performed every 24 hours: Agitationwas stopped to allow for sedimentation of the cell-containing carriers,and 80% of the culture supernatant was then harvested and replaced withnew medium. The harvested culture supernatant was filtered to removenon-trapped cells and cell debris and was then transferred for furtherprocessing. The 50 L as well as the 500 L bioreactor was instrumentedfor control of temperature, dissolved oxygen (sparging of oxygen throughmicrosparger), agitation rate, headspace aeration rate and pH (downwardscontrol by addition of CO₂ gas to headspace). Furthermore, the 500 Lbioreactor was instrumented for control of dissolved CO₂. Online CO₂measurement was performed by means of an YSI 8500 CO₂-instrument. Thelevel of CO₂ was controlled by sparging of atmospheric air into theliquid through a tube according to the CO₂ signal. The sparging rate wasset to 0 L/min per L of culture liquid when the CO₂ concentration was ator below the set-point, and to 0.01-0.05 L/min per L of culture liquidwhen the CO₂ concentration was above the set-point. The set-point fordissolved CO₂ was 160 mmHg. As mentioned, no base was added to thebioreactor to control pH upwards. During the production phase the celldensity reached 1-2×10⁷ cells/ml, and the Factor VII concentration inthe daily harvest 10-20 mg/L. The pCO₂ was maintained within the rangeof 150-170 mmHg. The pH was kept above 6.70, even though no base wasadded.

Example 2 Filtration of Eluate From the Capture Step

Protein solution to be filtered: 25 L of FVII solution from the capturestep, with the following characteristicsConcentration of FVII/FVIIa: 630 mg/L1.7% of oxidized forms of FVIIDegree of activation (i.e. percentage of FVIIa): not analysed

Degradation: <2.2%

The filtration was conducted essentially as described herein withreference to FIG. 1:

Filter: Millipore NFR, 0.08 m2 Pressure: 2 bar

Properties of the filtrate:Concentration of FVII/FVIIa: 610 mg/L, i.e.: yield of FVII: 96.8%1.5% of oxidized forms of FVIIDegree of activation (i.e. percentage of FVIIa): not analysed.

Degradation: <2.2% Example 3 Filtration of Eluate From the Capture Step

Protein solution to be filtered: 185 ml of FVII solution from thecapture step, with the following characteristicsConcentration of FVII/FVIIa: 82 mg/L3.4% of oxidized forms of FVIIDegree of activation (i.e. percentage of FVIIa): 19%.

Degradation: <3%

The filtration was conducted essentially as described herein withreference to FIG. 1:

Filter: Pall DV50, 0.0017 m2 Pressure: 2 bar

Properties of the filtrate:Concentration of FVII/FVIIa: 77.1 mg/L, i.e.: yield of FVII: 94%4.1% of oxidized forms of FVIIDegree of activation (i.e. percentage of FVIIa): 20%.

Degradation: <3% Example 4 Filtration of Eluate From the Capture Step

Protein solution to be filtered: 108 ml step 1 eluate with the followingcharacteristics:Concentration of FVII/FVIIa: 320 mg/L3.7% of oxidized forms of FVIIDegree of activation (i.e. percentage of FVIIa) 3.3%

Degradation: <0.5%

The filtration was conducted essentially as described herein withreference to FIG. 1:

Filter: Asahi Planova 20 N, 0.001 m2 Pressure: 0.8 bar

Properties of the filtrate:Concentration of FVII/FVIIa: 310 mg/L, i.e.: yield of FVII: 100%3.7% of oxidized forms of FVIIDegree of activation (i.e. percentage of FVIIa): not analysed,

Degradation: <0.5% Example 5 Filtration of FVII Bulk Drug Substance

Protein solution to be filtered: 98 ml of FVIIa bulk substance, with thefollowing characteristicsConcentration of FVII/FVIIa: 1460 mg/L2.1% of oxidized forms of FVIIDegree of activation (i.e. percentage of FVIIa): >90%.

Degradation: 11.9%

The filtration was conducted essentially as described herein withreference to FIG. 1:

Filter: Millipore NFP, 0.0017 m2 Pressure: 2 bar

Properties of the filtrate:Concentration of FVII/FVIIa: 1320 mg/L, i.e.: yield of FVII: 90.4%2.3% of oxidized forms of FVIIDegree of activation (i.e. percentage of FVIIa): not analysed, as thedegree of activation in the solution to be filtered is 98%

Degradation: 12.3% Example 6 Virus Removal

50 ml of a Factor VII polypeptide solution (see Example 1) from thecapture step comprising a Murine Leukemia Virus, titer YY plaque-formingunits (pfu).The filtration is conducted essentially as described herein withreference to FIG. 1:

Filter: Millipore NFR, yy cm2 Pressure: YY bar

Virus titer in the filtrate: xx pfuCalculated clearance factor: xx.

1. A method for removing viruses from a liquid composition ofrecombinant Factor VII comprising one or more Factor VII polypeptideshaving a concentration in the range of 0.01 to 5 mg/mL, the methodcomprising subjecting the liquid composition to nanofiltration using ananofilter having a pore size of 80 nm or less, wherein 50-100% of theFactor VII polypeptides in the composition subjected to the nanofilterare in an activated form (FVIIa) prior to nanofiltration.
 2. The methodaccording to claim 1, wherein the liquid composition has a pH of in therange of 7.0-9.5.
 3. The method according to claim 2, wherein the pH isin the range of 8.3-8.7.
 4. The method according to claim 1, wherein thepore size of the nanofilter is 50 nm or less.
 5. The method according toclaim 1, wherein the membrane of the nanofilter is manufactured from oneor more materials selected from the group consisting of: cuprammoniumregenerated cellulose, hydrophilic polyvinylidene fluoride (PVDF),composite PVDF, surface modified PVDF, and polyether sulfone.
 6. Themethod according to claim 1, wherein the pore size of the nanofilter isin the range of 10-30 nm.
 7. The method according to claim 1, whereinthe liquid Factor VII composition is obtained from a cell culturesupernatant.
 8. The method according to claim 1, wherein the liquidcomposition is serum-free.
 9. The method according to claim 1, whereinthe Factor VII polypeptide(s) is/are produced by cell culture in thepresence of bovine or foetal calf serum.
 10. The method according toclaim 1, wherein the Factor VII polypeptide(s) is/are produced by cellculture in Chinese Hamster Ovary (CHO) cells in a medium free from anycomponents of animal origin.
 11. The method according to claim 1,wherein the concentration of the Factor VII polypeptide(s) in the liquidcomposition is in the range of 0.05-2.0 mg/mL.
 12. A method for removingviruses from a liquid composition of recombinant Factor VII comprisingone or more Factor VII polypeptides having a concentration in the rangeof 0.01 to 5 mg/mL, wherein 50-100% of the Factor VII polypeptides inthe composition are in an activated form (FVIIa) prior tonanofiltration, the method comprising the steps of (i) combining thecomposition with a detergent, and (ii) subjecting the solution tonanofiltration using a nanofilter having a pore size of 80 nm or less.13. The method of claim 1, wherein 70-100% of the Factor VIIpolypeptides in the composition subjected to the nanofilter are theactivated form of Factor VII.
 14. The method of claim 1, wherein 80-100%of the Factor VII polypeptides in the composition subjected to thenanofilter are the activated form of Factor VII.
 15. A method forremoving viruses from a liquid composition of recombinant Factor VIIcomprising one or more Factor VII polypeptides having a concentration inthe range of 0.01 to 5 mg/mL, the method comprising subjecting theliquid composition to nanofiltration using a nanofilter having a poresize of 50 nm or less, wherein 50-100% of the Factor VII polypeptides inthe composition subjected to the nanofilter are in an activated form(FVIIa) prior to nanofiltration.